Detection of nucleic acids by target-catalyzed product formation

ABSTRACT

A method is disclosed for modifying an oligonucleotide, which method has application to the detection of a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide, for example, a polynucleotide analyte, in the presence of a 5′-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5′-nuclease to cleave the oligonucleotide to provide (i) a first fragment-that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3′ of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte. The method has particular application to the detection of a polynucleotide analyte such as DNA. Kits for conducting methods in accordance with the present invention are also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Nucleic acid hybridization has been employed for investigating theidentity and establishing the presence of nucleic acids. Hybridizationis based on complementary base pairing. When complementary singlestranded nucleic acids are incubated together, the complementary basesequences pair to form double stranded hybrid molecules. The ability ofsingle stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA)to form a hydrogen bonded structure with a complementary nucleic acidsequence has been employed as an analytical tool in molecular biologyresearch. The availability of radioactive nucleoside triphosphates ofhigh specific activity and the 32P labelling of DNA with T4polynucleotide kinase has made it possible to identify, isolate, andcharacterize various nucleic acid sequences of biological interest.Nucleic acid hybridization has great potential in diagnosing diseasestates associated with unique nucleic acid sequences. These uniquenucleic acid sequences may result from genetic or environmental changein DNA by insertions, deletions, point mutations, or by acquiringforeign DNA or RNA by means of infection by bacteria, molds, fungi, andviruses. Nucleic acid hybridization has, until now, been employedprimarily in academic and industrial molecular biology laboratories. Theapplication of nucleic acid hybridization as a diagnostic tool inclinical medicine is limited because of the frequently very lowconcentrations of disease related DNA or RNA present in a patient's bodyfluid and the unavailability of a sufficiently sensitive method ofnucleic acid hybridization analysis.

Current methods for detecting specific nucleic acid sequences generallyinvolve immobilization of the target nucleic acid on a solid supportsuch as nitrocellulose paper, cellulose paper, diazotized paper, or anylon membrane. After the target nucleic acid is fixed on the support,the support is contacted with a suitably labelled probe nucleic acid forabout two to forty-eight hours. After the above time period, the solidsupport is washed several times at a controlled temperature to removeunhybridized probe. The support is then dried and the hybridizedmaterial is detected by autoradiography or by spectrometric methods.

When very low concentrations must be detected, the current methods areslow and labor intensive, and nonisotopic labels that are less readilydetected than radiolabels are frequently not suitable. A method forincreasing the sensitivity to permit the use of simple, rapid,nonisotopic, homogeneous or heterogeneous methods for detecting nucleicacid sequences is therefore desirable.

Recently, a method for the enzymatic amplification of specific segmentsof DNA known as the polymerase chain reaction (PCR) method has beendescribed. This in vitro amplification procedure uses two or moredifferent oligonucleotide primers for different strands of the targetnucleic acid and is based on repeated cycles of denaturation,oligonucleotide primer annealing, and primer extension by thermophilicpolymerase, resulting in the exponential increase in copies of theregion flanked by the primers. The different PCR primers, which annealto opposite strands of the DNA, are positioned so that the polymerasecatalyzed extension product of one primer can serve as a template strandfor the other primer, leading to the accumulation of discrete fragmentswhose length is defined by the distance between the 5′-ends of theoligonucleotide primers.

Other methods for amplifying nucleic acids are single primeramplification, ligase chain reaction (LCR), nucleic acid sequence basedamplification (NASBA) and the Q-beta-replicase method. Regardless of theamplification used, the amplified product must be detected.

Depending on which of the above amplification methods are employed, themethods generally employ from seven to twelve or more reagents.Furthermore, the above methods provide for exponential amplification ofa target or a reporter oligonucleotide. Accordingly, it is necessary torigorously avoid contamination of assay solutions by the amplifiedproducts to avoid false positives. Some of the above methods requireexpensive thermal cycling instrumentation and additional reagents andsample handling steps are needed for detection of the amplified product.

Most assay methods that do not incorporate amplification of a target DNAavoid the problem of contamination, but they are not adequatelysensitive or simple. Some of the methods involve some type of sizediscrimination such as electrophoresis, which adds to the complexity ofthe methods.

One method for detecting nucleic acids is to employ nucleic acid probes.One method utilizing such probes is described in U.S. Pat. No.4,868,104, the disclosure of which is incorporated herein by reference.A nucleic acid probe may be, or may be capable of being, labeled with areporter group or may be, or may be capable of becoming, bound to asupport.

Detection of signal depends upon the nature of the label or reportergroup. If the label or reporter group is an enzyme, additional membersof the signal producing system include enzyme substrates and so forth.The product of the enzyme reaction is preferably a luminescent product,or a fluorescent or non-fluorescent dye, any of which can be detectedspectrophotometrically, or a product that can be detected by otherspectrometric or electrometric means. If the label is a fluorescentmolecule, the medium can be irradiated and the fluorescence determined.Where the label is a radioactive group, the medium can be counted todetermine the radioactive count.

It is desirable to have a sensitive, simple method for detecting nucleicacids. The method should minimize the number and complexity of steps andreagents. The need for sterilization and other steps needed to preventcontamination of assay mixtures should be avoided.

2. Description of the Related Art

Methods for detecting nucleic acid sequences are discussed by Duck, etal., in U.S. Pat. No. 5,011,769 and corresponding International PatentApplication WO 89/10415. A method of cleaving a nucleic acid molecule isdisclosed in European Patent Application 0 601 834 A1 (Dahlberg, etal.).

Holland, et al., Clinical Chemistry (1992) 38:462-463, describedetection of specific polymerase chain reaction product by utilizing the5′ to 3′ exonuclease activity of Thermus aguaticus DNA polymerase.Longley, et al., Nucleic Acids Research (1990) 18:7317-7322, discusscharacterization of the 5′ to 3′ exonuclease associated with Thermusaguaticus DNA polymerase. Lyamichev, et al., Science (1993) 260:778-783,disclose structure-specific endonucleolytic cleavage of nucleic acids byeubacterial DNA polymerases.

A process for amplifying, detecting and/or cloning nucleic acidsequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159, 4,965,188 and 5,008,182. Sequence polymerization bypolymerase chain reaction is described by Saiki, et al., (1986) Science,230: 1350-1354. Primer-directed enzymatic amplification of DNA with athermostable DNA polymerase is described by Saiki, et al., Science(1988) 239:487.

U.S. patent applications Ser. Nos. 07/299,282 and 07/399,795, filed Jan.19, 1989, and Aug. 29, 1989, respectively, describe nucleic acidamplification using a single polynucleotide primer. The disclosures ofthese applications are incorporated herein by reference including thereferences listed in the sections entitled “Description of the RelatedArt.”

Other methods of achieving the result of a nucleic acid amplificationare described by Van Brunt in Bio/Technology (1990) 8(No.4): 291-294.These methods include ligase chain reaction (LCR), nucleic acid sequencebased amplification (NASBA) and Q-beta-replicase amplification of RNA.LCR is also discussed in European Patent Applications Nos. 439,182(Backman I) and 473,155 (Backman II).

NASBA is a promoter-directed, isothermal enzymatic process that inducesin vitro continuous, homogeneous and isothermal amplification ofspecific nucleic acid.

Q-beta-replicase relies on the ability of Q-beta-replicase to amplifyits RNA substrate exponentially under isothermal conditions.

Another method for conducting an amplification of nucleic acids isreferred to as strand displacement amplification (SDA). SDA is anisothermal, in vitro DNA amplification technique based on the ability ofa restriction enzyme to nick the unmodified strand of ahemiphosphorothioate form of its restriction site and the ability of aDNA polymerase to initiate replication at the nick and displace thedownstream nontemplate strand intact. Primers containing the recognitionsites for the nicking restriction enzyme drive the exponentialamplification.

Another amplification procedure for amplifying nucleic acids is known as3SR, which is an RNA specific target method whereby RNA is amplified inan isothermal process combining promoter directed RNA polymerase,reverse transcriptase and RNase H with target RNA.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method for modifying anoligonucleotide. The method comprises incubating the oligonucleotidewith a polynucleotide and a 5′-nuclease wherein at least a portion ofthe oligonucleotide-is reversibly hybridized to the polynucleotide underisothermal conditions. The oligonucleotide is cleaved to provide (i) afirst fragment that is substantially non-hybridizable to thepolynucleotide and includes no more than one nucleotide from the 5′-endof the portion and (ii) a second fragment that is 3′ of the firstfragment with reference to the intact oligonucleotide and issubstantially hybridizable to the polynucleotide.

Another aspect of the present invention is a method for detecting apolynucleotide analyte. An oligonucleotide is reversibly hybridized witha polynucleotide analyte and a 5′-nuclease under isothermal conditions.The polynucleotide analyte serves as a recognition element to enable a5′-nuclease to cleave the oligonucleotide to provide (i) a firstfragment that is substantially non-hybridizable to the polynucleotideanalyte and (ii) a second fragment that lies 3′ of the first fragment(in the intact oligonucleotide) and is substantially hybridizable to thepolynucleotide analyte. At least a 100-fold molar excess of the firstfragment and/or the second fragment are obtained relative to the molaramount of the polynucleotide is analyte. The presence of the firstfragment and/or the second fragment is detected, the presence thereofindicating the presence of the polynucleotide analyte.

Another embodiment of the present invention is a method for detecting apolynucleotide analyte. A combination is provided comprising a mediumsuspected of containing the polynucleotide analyte, an excess, relativeto the suspected concentration of the polynucleotide analyte, of a firstoligonucleotide at least apportion of which is capable of reversiblyhybridizing with the polynucleotide analyte under isothermal conditions,a 5′-nuclease, and a second oligonucleotide having the characteristic ofhybridizing to a site on the polynucleotide analyte that is 3′ of thesite at which the first oligonucleotide hybridizes. The polynucleotideanalyte is substantially fully hybridized to the second oligonucleotideunder such isothermal conditions. The polynucleotide is reversiblyhybridized under the isothermal conditions to the first oligonucleotide,which is cleaved as a function of the presence of the polynucleotideanalyte to provide, in at least a 100-fold molar excess of thepolynucleotide analyte, (i) a first fragment that is substantiallynon-hybridizable to the polynucleotide analyte and/or (ii) a secondfragment that lies 3′ of the first fragment (in the intact firstoligonucleotide) and is substantially hybridizable to the polynucleotideanalyte. The presence of the first fragment and/or the second fragmentis detected, the presence thereof indicating the presence of thepolynucleotide analyte.

Another embodiment of the present invention is a method for detecting aDNA analyte. A combination is provided comprising a medium suspected ofcontaining the DNA analyte, a first oligonucleotide at least a portionof which is capable of reversibly hybridizing with the DNA analyte underisothermal conditions, a 5′-nuclease, and a second oligonucleotidehaving the characteristic of hybridizing to a site on the DNA analytethat is 3′ of the site at which the first oligonucleotide hybridizes.The DNA analyte is substantially fully hybridized to the secondoligonucleotide under isothermal conditions. The polynucleotide analyteis reversibly hybridized to the first oligonucleotide under isothermalconditions. The first oligonucleotide is cleaved to (i) a first fragmentthat is substantially non-hybridizable to the DNA analyte and (ii) asecond fragment that lies 3′ of the first fragment (in the intact firstoligonucleotide) and is substantially hybridizable to the DNA analyte.At least a 100-fold molar excess, relative to the DNA analyte, of thefirst fragment and/or the second fragment is produced. The presence ofthe first fragment and/or the second fragment is detected, the presencethereof indicating the presence of the DNA analyte.

Another embodiment of the present invention is a kit for detection of apolynucleotide. The kit comprises in packaged combination (a) a firstoligonucleotide having the characteristic that, when reversiblyhybridized under isothermal conditions to the polynucleotide, it isdegraded by a 5′-nuclease to provide (i) a first fragment that issubstantially non-hybridizable to the polynucleotide and (ii) a secondfragment that is 3′ of the first fragment (in the first oligonucleotide)and is substantially hybridizable to the polynucleotide, (b) a secondoligonucleotide having the characteristic of hybridizing to a site onthe polynucleotide that is separated by no more than one nucleotide fromthe 3′-end of the site at which the first oligonucleotide hybridizeswherein the polynucleotide is substantially fully hybridized to thesecond oligonucleotide under the isothermal conditions, and (c) a5′-nuclease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 are schematics of different embodiments in accordance with thepresent invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention permits catalyzed cleavage of an oligonucleotidethat is modulated by a portion of a polynucleotide analyte, such as apolynucleotide, that is comprised of a target polynucleotide sequence towhich a portion of the oligonucleotide hybridizes. As such, the methodsof the present invention provide for very high sensitivity assays forpolynucleotide analytes. The methods are simple to conduct and notemperature cycling is required. Consequently, no expensive thermalcycling instrumentation is needed. Furthermore, only a few reagents areused, thus further minimizing cost and complexity of an assay. Inaddition, the absence of amplified products, which are potentialamplification targets, permits the use of less rigorous means to avoidcontamination of assay solutions by target sequences that could producefalse positives.

Before proceeding further with a description of the specific embodimentsof the present invention, a number of terms will be defined.

Polynucleotide analyte—a compound or composition to be measured that isa polymeric nucleotide, which in the intact natural state can have about20 to 500,000 or more nucleotides and in an isolated state can haveabout 30 to 50,000 or more nucleotides, usually about 100 to 20,000nucleotides, more frequently 500 to 10,000 nucleotides. Isolation ofanalytes from the natural state, particularly those having a largenumber of nucleotides, frequently results in fragmentation. Thepolynucleotide analytes include nucleic acids from any source inpurified or unpurified form including DNA (dsDNA and ssDNA) and RNA,including t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplastDNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, chromosomes,plasmids, the genomes of biological material such as microorganisms,e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, animals,humans, and fragments thereof, and the like. Preferred polynucleotideanalytes are double stranded DNA (dsDNA) and single stranded DNA(ssDNA). The polynucleotide analyte can be only a minor fraction of acomplex mixture such as a biological sample. The analyte can be obtainedfrom various biological material by procedures well known in the art.Some examples of such biological material by way of illustration and notlimitation are disclosed in Table I below. TABLE I Microorganisms ofinterest include: Corynebacteria Corynebacterium diphtheria PneumococciDiplococcus pneumoniae Streptococci Streptococcus pyrogenesStreptococcus salivarus Staphylococci Staphylococcus aureusStaphylococcus albus Neisseria Neisseria meningitidis Neisseriagonorrhea Enterobacteriaciae Escherichia coli Aerobacter aerogenes Thecolliform Klebsiella pneumoniae bacteria Salmonella typhosa Salmonellacholeraesuis The Salmonellae Salmonella typhimurium Shigella dysenteriaShigella schmitzii Shigella arabinotarda The Shigellae Shigella flexneriShigella boydii Shigella sonnei Other enteric bacilli Proteus vulgarisProteus mirabilis Proteus species Proteus morgani Pseudomonas aeruginosaAlcaligenes faecalis Vibrio cholerae Hemophilus-Bordetella qroupRhizopus oryzae Hemophilus influenza, H. ducryi Rhizopus arrhizuaPhycomycetes Hemophilus hemophilus Rhizopus nigricans Hemophilusaegypticus Sporotrichum schenkii Hemophilus parainfluenza Flonsecaeapedrosoi Bordetella pertussis Fonsecacea compact Pasteurellae Fonsecaceadermatidis Pasteurella pestis Cladosporium carrionii Pasteurellatulareusis Phialophora verrucosa Brucellae Aspergillus nidulans Brucellamelitensis Madurella mycetomi Brucella ahortus Madurella grisea Brucellasuis Allescheria boydii Aerobic Spore-forming Bacilli Phialophorajeanselmei Bacillus anthracis Microsporum gypseum Bacillus subtilisTrichophyton mentagrophytes Bacillus megaterium Keratinomyces ajelloiBacillus cereus Microsporum canis Anaerobic Spore-forming BacilliTrichophyton rubrum Clostridium botulinum Microsporum adouiniClostridium tetani Viruses Clostridium perfringens AdenovirusesClostridium novyi Herpes Viruses Clostridium septicum Herpes simplexClostridium histolyticum Varicella (Chicken pox) Clostridium tertiumHerpes Zoster (Shingles) Clostridium bifermentans Virus B Clostridiumsporogenes Cytomegalovirus Mycobacteria Pox viruses Mycobacteriumtuberculosis Variola (smallpox) hominis Mycobacterium bovis VacciniaMycobacterium avium Poxvirus bovis Mycobacterium leprae ParavacciniaMycobacterium paratuberculosis Molluscum contagiosum Actinomycetes(fungus-like bacteria) Picornaviruses Actinomyces Isaeli PoliovirusActinomyces bovis Coxsackievirus Actinomyces naeslundii EchovirusesNocardia asteroides Rhinoviruses Nocardia brasiliensis Myxoviruses TheSpirochetes Influenza (A, B, and C) Treponema pallidum Spirillum minusParainfluenza (1-4) Treponema pertenue Streptobacillus Mumps Virusmonoiliformis Newcastle Disease Virus Treponema carateum Measles VirusBorrelia recurrentis Rinderpest Virus Leptospira icterohemorrhagiaeCanine Distemper Virus Leptospira canicola Respiratory Syncytial VirusTrypanasomes Rubella Virus Mycoplasmas Arboviruses Mycoplasma pneumoniaeOther pathogens Eastern Equine Encephalitis Virus Listeria monocytogenesWestern Equine Encephalitis Virus Erysipelothrix rhusiopathiae SindbisVirus Streptobacillus moniliformis Chikugunya Virus Donvaniagranulomatis Semliki Forest Virus Bartonella bacilliformis Mayora VirusRickettsiae (bacteria-like St. Louis parasites) Encephalitis VirusRickettsia prowazekii California Encephalitis Virus Rickettsia mooseriColorado Tick Fever Virus Rickettsia rickettsii Yellow Fever VirusRickettsia conori Dengue Virus Rickettsia australis ReovirusesRickettsia sibiricus Reovirus Types 1-3 Retroviruses Rickettsia akariHuman (HIV) Immunodeficiency Viruses Rickettsia tsutsugamushi HumanT-cell Lymphotrophic Virus I & II (HTLV) Rickettsia burnetti HepatitisRickettsia quintana Hepatitis A Virus Chlamydia (unclassifiableparasites Hepatitis B Virus bacterial/viral) Hepatitis nonA- nonB VirusChlamydia agents (naming uncertain) Tumor Viruses Fungi RauscherLeukemia Virus Cryptococcus neoformans Gross Virus Blastomycesdermatidis Maloney Leukemia Virus Hisoplasma capsulatum Coccidioidesimmitis Human Papilloma Virus Paracoccidioides brasiliensis Candidaalbicans Aspergillus fumigatus Mucor corymbifer (Absidia corymbifera)

The polynucleotide analyte, where appropriate, may be treated to cleavethe analyte to obtain a polynucleotide that contains a targetpolynucleotide sequence, for example, by shearing or by treatment with arestriction endonuclease or other site specific chemical cleavagemethod. However, it is an advantage of the present invention that thepolynucleotide analyte can be used in its isolated state without furthercleavage.

For purposes of this invention, the polynucleotide analyte, or a cleavedpolynucleotide obtained from the polynucleotide analyte, will usually beat least partially denatured or single stranded or treated to render itdenatured or single stranded. Such treatments are well-known in the artand include, for instance, heat or alkali treatment. For example, doublestranded DNA can be heated at 90-100° C. for a period of about 1 to 10minutes to produce denatured material.

3′- or 5′-End of an oligonucleotide—as used herein this phrase refers toa portion of an oligonucleotide comprising the 3′- or 5′-terminus,respectively, of the oligonucleotide.

3′- or 5′-Terminus of an oligonucleotide—as used herein this term refersto the terminal nucleotide at the 3′- or 5′-end, respectively, of anoligonucleotide.

Target polynucleotide sequence—a sequence of nucleotides to beidentified, which may be the polynucleotide analyte but is usuallyexisting within a polynucleotide comprising the polynucleotide analyte.The identity of the target polynucleotide sequence is known to an extentsufficient to allow preparation of an oligonucleotide having a portionor sequence that hybridizes with the target polynucleotide sequence. Ingeneral, when one oligonucleotide is used, the oligonucleotidehybridizes with the 5′-end of the target polynucleotide sequence. When asecond oligonucleotide is used, it hybridizes to a site on the targetpolynucleotide sequence that is 3′ of the site to which the firstoligonucleotide hybridizes. (It should be noted that the relationshipcan be considered with respect to the double stranded molecule formedwhen the first and second oligonucleotides are hybridized to thepolynucleotide. In such context the second oligonucleotide is5-primeward of the first oligonucleotide with respect to the “strand”comprising the first and second oligonucleotides.) The relationshipsdescribed above are more clearly seen with reference to FIG. 3. Thetarget polynucleotide sequence usually contains from about 10 to 1,000nucleotides, preferably 15 to 100 nucleotides, more preferably, 20 to 70nucleotides. The target polynucleotide sequence is part of apolynucleotide that may be the entire polynucleotide analyte. Theminimum number of nucleotides in the target polynucleotide sequence isselected to assure that the presence of target polynucleotide sequencein a sample is a specific indicator of the presence of polynucleotideanalyte in a sample. Very roughly, the sequence length is usuallygreater than about 1.6 log L nucleotides where L is the number of basepairs in the genome of the biologic source of the sample. The number ofnucleotides in the target sequence is usually the sum of the lengths ofthose portions of the oligonucleotides that hybridize with the targetsequence plus the number of nucleotides lying between the portions ofthe target sequence that hybridize with the oligonucleotides.

Oligonucleotide—a polynucleotide, usually a synthetic polynucleotide,usually single stranded that is constructed such that at least a portionthereof hybridizes with the target polynucleotide sequence of thepolynucleotide. The oligonucleotides of this invention are usually 10 to150 nucleotides, preferably, deoxyoligonucleotides of 15 to 100nucleotides, more preferably, 20 to 60 nucleotides, in length.

The first oligonucleotide, or “the” oligonucleotide when a secondoligonucleotide is not employed, has a 5′-end about 0 to 100nucleotides, preferably, 1 to 20 nucleotides in length that does nothybridize with the target polynucleotide sequence and usually has a 10to 40 nucleotide sequence that hybridizes with the target polynucleotidesequence. In general, the degree of amplification is reduced somewhat asthe length of the portion of the oligonucleotide that does not hybridizewith the target polynucleotide sequence increases. The firstoligonucleotide also may have a sequence at its 3′-end that does nothybridize with the target polynucleotide sequence.

The second oligonucleotide preferably hybridizes at its 3′-end with thetarget polynucleotide sequence at a site on the target polynucleotidesequence 3′ of the site of binding of the first oligonucleotide. Thelength of the portion of the second oligonucleotide that hybridizes withthe target polynucleotide sequence is usually longer than the length ofthe portion of the first oligonucleotide that hybridizes with the targetpolynucleotide sequence and is usually 20 to 100 nucleotides. Themelting temperature of the second oligonucleotide hybridized to thetarget polynucleotide sequence is preferably at least as high, morepreferably, at least 50° C. higher than the melting temperature of thefirst oligonucleotide hybridized to the target polynucleotide sequence.

The oligonucleotides can be oligonucleotide mimics such apolynucleopeptides, phosphorothioates or phosphonates except that thefirst oligonucleotide usually has at least one phosphodiester bond tothe nucleoside at the 5′-end of the sequence that hybridizes with thetarget polynucleotide sequence. When oligonucleotide mimics are usedthat provide very strong binding, such as polynucleopeptides, the lengthof the portion of the second oligonucleotide that hybridizes with thetarget polynucleotide sequence may be reduced to less than 20 and,preferably, greater than 10.

Various techniques can be employed for preparing an oligonucleotide orother polynucleotide utilized in the present invention. They can beobtained by biological synthesis or by chemical synthesis. For shortoligonucleotides (up to about 100 nucleotides) chemical synthesis willfrequently be more economical as compared to biological synthesis. Inaddition to economy, chemical synthesis provides a convenient way ofincorporating low molecular weight compounds and/or modified basesduring the synthesis step. Furthermore, chemical synthesis is veryflexible in the choice of length and region of the target polynucleotidesequence. The oligonucleotides can be synthesized by standard methodssuch as those used in commercial automated nucleic acid synthesizers.Chemical synthesis of DNA on a suitably modified glass or resin resultsin DNA covalently attached to the surface. This may offer advantages inwashing and sample handling. For longer sequences standard replicationmethods employed in molecular biology can be used such as the use of M13for single stranded DNA as described by J. Messing (1983) MethodsEnzymol, 101, 20-78.

In addition to standard cloning techniques, in vitro enzymatic methodsmay be used such as polymerase catalyzed reactions. For preparation ofRNA, T7 RNA polymerase and a suitable DNA template can be used. For DNA,polymerase chain reaction (PCR) and single primer amplification areconvenient.

Other chemical methods of polynucleotide or oligonucleotide synthesisinclude phosphotriester and phosphodiester methods (Narang, et al.,Meth. Enzymol (1979) 68: 90) and synthesis on a support (Beaucage, etal., Tetrahedron (1981) Letters 22: 1859-1862) as well asphosphoramidate techniques, Caruthers, M. H., et al., “Methods inEnzymology,” Vol. 154, pp. 287-314 (1988), and others described in“Synthesis and Applications of DNA and RNA,” S. A. Narang, editor,Academic Press, New York, 1987, and the references contained therein.

Fragment—in general, in the present method the oligonucleotide (or thefirst oligonucleotide when a second oligonucleotide is employed) iscleaved only when at least a portion thereof is reversibly hybridizedwith a target polynucleotide sequence and, thus, the targetpolynucleotide sequence acts as a recognition element for cleavage ofthe oligonucleotide, thereby yielding two portions. One fragment issubstantially non-hybridizable to the target polynucleotide sequence.The other fragment is substantially hybridizable to the targetpolynucleotide sequence and 3′ of the other fragment with respect to theoligonucleotide in its uncleaved form.

5′-Nuclease—a sequence-independent deoxyribonuclease enzyme thatcatalyzes the cleavage of an oligonucleotide into fragments only when atleast a portion of the oligonucleotide is hybridized to the targetpolynucleotide sequence. The enzyme selectively cleaves theoligonucleotide near the 5′-terminus of the bound portion, within 5nucleotides thereof, preferably within 1 to 2 nucleotides thereof anddoes not cleave the unhybridized oligonucleotide or the targetpolynucleotide sequence. Such enzymes include both 5′-exonucleases and5′-endonucleases but exclude ribonucleases such as RNAse H andrestriction enzymes. 5′-nucleases useful in the present invention mustbe stable under the isothermal conditions used in the present method andare usually thermally stable nucleotide polymerases having5′-exonuclease activity such as Taq DNA polymerase (e.g. AmpliTaq(™)from Perkin-Elmer Corporation, Norwalk, N.J.), Thermalase Tbr(™) DNApolymerase (from Amresco, Solon, Ohio), Ultra Therm(™) DNA polymerase(from Bio/Can Scientific, Ontario, Canada), Replitherm(™) DNA polymerase(from Epicentre, Madison, Wis.), Tfl(™) DNA polymerase (from Epicentre),Panozyme(™) DNA polymerase (from Panorama Research, Mountain View,Calif.), Tth(™) DNA polymerase (from Epicentre), rBst(™) DNA polymerase(from Epicentre), Heat Tuff(™) DNA polymerase (from Clontech, Palo Alto,Calif.), and the like, derived from any source such as cells, bacteria,such as E. coli, plants, animals, virus, thermophilic bacteria, and soforth wherein the polymerase may be modified chemically or throughgenetic engineering to provide for thermal stability and/or increasedactivity.

Isothermal conditions—a uniform or constant temperature at which themodification of the oligonucleotide in accordance with the presentinvention is carried out. The temperature is chosen so that the duplexformed by hybridizing the oligonucleotide to a polynucleotide with atarget polynucleotide sequence is in equilibrium with the free orunhybridized oligonucleotide and free or unhybridized targetpolynucleotide sequence, a condition that is otherwise referred toherein as “reversibly hybridizing” the oligonucleotide with apolynucleotide. Normally, at least 1%, preferably 20 to 80%, usuallyless than 95% of the polynucleotide is hybridized to the oligonucleotideunder the isotermal conditions. Accordingly, under isothermal conditionsthere are molecules of polynucleotide that are hybridized with theoligonucleotide, or portions thereof, and are in dynamic equilibriumwith molecules that are not hybridized with the oligonucleotide. Somefluctuation of the temperature may occur and still achieve the benefitsof the present invention. The fluctuation generally is not necessary forcarrying out the methods of the present invention and usually offer nosubstantial improvement. Accordingly, the term “isothermal conditions”includes the use of a fluctuating temperature, particularly random oruncontrolled fluctuations in temperature, but specifically excludes thetype of fluctuation in temperature referred to as thermal cycling, whichis employed in some known amplification procedures, e.g., polymerasechain reaction.

Polynucleotide primer(s) or oligonucleotide primer(s)—an oligonucleotidethat is usually employed in a chain extension on a polynucleotidetemplate.

Nucleoside triphosphates—nucleosides having a 5′-triphosphatesubstituent. The nucleosides are pentose sugar derivatives ofnitrogenous bases of either purine or pyrimidine derivation, covalentlybonded to the 1′-carbon of the pentose sugar, which is usually adeoxyribose or a ribose. The purine bases include adenine(A),guanine(G), inosine, and derivatives and analogs thereof. The pyrimidinebases include cytosine (C), thymine (T), uracil (U), and derivatives andanalogs thereof. Nucleoside triphosphates include deoxyribonucleosidetriphosphates such as DATP, dCTP, dGTP and dTTP and ribonucleosidetriphosphates such as rATP, rCTP, rGTP and rUTP. The term “nucleosidetriphosphates” also includes derivatives and analogs thereof.

Nucleotide—a base-sugar-phosphate combination that is the monomeric unitof nucleic acid polymers, i.e., DNA and RNA.

Nucleoside—is a base-sugar combination or a nucleotide lacking aphosphate moiety.

Nucleotide polymerase—a catalyst, usually an enzyme, for forming anextension of an oligonucleotide along a polynucleotide template wherethe extension is complementary thereto. The nucleotide polymerase is atemplate dependent polynucleotide polymerase and utilizes nucleosidetriphosphates as building blocks for extending the 3′-end of aoligonucleotide to provide a sequence complementary with the singlestranded portion of the polynucleotide to which the oligonucleotide ishybridized to form a duplex.

Hybridization (hybridizing) and binding—in the context of nucleotidesequences these terms are used interchangeably herein. The ability oftwo nucleotide sequences to hybridize with each other is based on thedegree of complementarity of the two nucleotide sequences, which in turnis based on the fraction of matched complementary nucleotide pairs. Themore nucleotides in a given sequence that are complementary to anothersequence, the more stringent the conditions can be for hybridization andthe more specific will be the binding of the two sequences. Increasedstringency is achieved by elevating the temperature, increasing theratio of cosolvents, lowering the salt concentration, and the like.

Homologous or substantially identical—In general, two polynucleotidesequences that are identical or can each hybridize to the samepolynucleotide sequence are homologous. The two sequences are homologousor substantially identical where the sequences each have at least 90%,preferably 100%, of the same or analogous base sequence where thymine(T) and uracil (U) are considered the same. Thus, the ribonucleotides A,U, C and G are taken as analogous to the deoxynucleotides dA, dT, dC,and dG, respectively. Homologous sequences can both be DNA or one can beDNA and the other RNA.

Complementary—Two sequences are complementary when the sequence of onecan bind to the sequence of the other in an anti-parallel sense whereinthe 3′-end of each sequence binds to the 5′-end of the other sequenceand each A, T(U), G, and C of one sequence is then aligned with a T(U),A, C, and G, respectively, of the other sequence.

Copy—means a sequence that is a direct identical or homologous copy of asingle stranded polynucleotide sequence as differentiated from asequence that is complementary to the sequence of such single strandedpolynucleotide.

Member of a specific binding pair (“sbp member”)—one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to, and is thereby defined as complementary with, aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These may be members of an immunological pairsuch as antigen-antibody, or may be operator-repressor,nuclease-nucleotide, biotin-avidin, hormones-hormone receptors, nucleicacid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like.

Ligand—any compound for which a receptor naturally exists or can beprepared.

Receptor (“antiligand”)—any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors,protection enzymes, protein A, complement component Clq, DNA bindingproteins or ligands and the like.

Small organic molecule—a compound of molecular weight less than 1500,preferably 100 to 1000, more preferably 300 to 600 such as biotin,fluorescein, rhodamine and other dyes, tetracycline and other proteinbinding molecules, and haptens, etc. The small organic molecule canprovide a means for attachment of a nucleotide sequence to a label or toa support or may itself be a label.

Support or surface—a porous or non-porous water insoluble material. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

Binding of sbp members to a support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface canhave any one of a number of shapes, such as strip, rod, particle,including bead, and the like.

Label or reporter group or reporter molecule—a member of a signalproducing system. Usually the label or reporter group or reportermolecule is conjugated to or becomes bound to, or fragmented from, anoligonucleotide or to a nucleoside triphosphate and is capable of beingdetected directly or, through a specific binding reaction, and canproduce a detectible signal. In general, any label that is detectablecan be used. The label can be isotopic or nonisotopic, usuallynon-isotopic, and can be a catalyst, such as an enzyme or a catalyticpolynucleotide, promoter, dye, fluorescent molecule, chemiluminescer,coenzyme, enzyme substrate, radioactive group, a small organic molecule,amplifiable polynucleotide sequence, a particle such as latex or carbonparticle, metal sol, crystallite, liposome, cell, etc., which may or maynot be further labeled with a dye, catalyst or other detectible group,and the like. Labels include an oligonucleotide or specificpolynucleotide sequence that can provide a template for amplification orligation or act as a ligand such as for a repressor protein. The labelis a member of a signal producing system and can generate a detectablesignal either alone or together with other members of the signalproducing system. The label can be bound directly to a nucleotidesequence or can become bound thereto by being bound to an sbp membercomplementary to an sbp member that is bound to a nucleotide sequence.

Signal Producing System—The signal producing system may have one or morecomponents, at least one component being the label or reporter group orreporter molecule. The signal producing system generates a signal thatrelates to the presence or amount of target polynucleotide sequence or apolynucleotide analyte in a sample. The signal producing system includesall of the reagents required to produce a measurable signal. When thelabel is not conjugated to a nucleotide sequence, the label is normallybound to an sbp member complementary to an sbp member that is bound to,or part of, a nucleotide sequence. Other components of the signalproducing system may be included in a developer solution and can includesubstrates, enhancers, activators, chemiluminescent compounds,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Other components of the signal producing system may be coenzymes,substances that react with enzymic products, other enzymes andcatalysts, and the like. The signal producing system provides a signaldetectable by external means, by use of electromagnetic radiation,desirably by visual examination. The signal-producing system isdescribed more fully in U.S. patent application Ser. No. 07/555,323,filed Jul. 19, 1990, the relevant disclosure of which is incorporatedherein by reference.

Amplification of nucleic acids or polynucleotides—any method thatresults in the formation of one or more copies of a nucleic acid or apolynucleotide molecule, usually a nucleic acid or polynucleotideanalyte, or complements thereof, present in a medium.

Exponential amplification of nucleic acids or polynucleotides—any methodthat results in the formation of one or more copies of a nucleic acid orpolynucleotide molecule, usually a nucleic acid or polynucleotideanalyte, present in a medium.

Methods for the enzymatic amplification of specific double strandedsequences of DNA include those described above such as the polymerasechain reaction (PCR), amplification of a single stranded polynucleotideusing a single polynucleotide primer, ligase chain reaction (LCR),nucleic acid sequence based amplification (NASBA), Q-beta-replicasemethod, strand displacement amplification (SDA), and 3SR.

Conditions for carrying out an amplification, thus, vary depending uponwhich method is selected. Some of the methods such as PCR utilizetemperature cycling to achieve denaturation of duplexes, oligonucleotideprimer annealing, and primer extension by thermophilic templatedependent polynucleotide polymerase. Other methods such as NASBA,Q-beta-replicase method, SDA and 3SR are isothermal. As can be seen,there are a variety of known amplification methods and a variety ofconditions under which these methods are conducted to achieveexponential amplification.

Linear amplification of nucleic acids or polynucleotides—any method thatresults in the formation of one or more copies of only the complement ofa nucleic acid or polynucleotide molecule, usually a nucleic acid orpolynucleotide analyte, present in a medium. Thus, one differencebetween linear amplification and exponential amplification is that thelatter produces copies of the polynucleotide whereas the former producesonly the complementary strand of the polynucleotide. In linearamplification the number of complements formed is, in principle,directly proportional to the time of the reaction as opposed toexponential amplification wherein the number of copies is, in principle,an exponential function of the time or the number of temperature cycles.

Ancillary Materials—various ancillary materials will frequently beemployed in the methods and assays carried out in accordance with thepresent invention. For example, buffers will normally be present in theassay medium, as well as stabilizers for the assay medium and the assaycomponents. Frequently, in addition to these additives, proteins may beincluded, such as albumins, organic solvents such as formamide,quaternary ammonium salts, polycations such as dextran sulfate,surfactants, particularly non-ionic surfactants, binding enhancers,e.g., polyalkylene glycols, or the like.

As mentioned above, the present invention has a primary application tomethods for detecting a polynucleotide analyte. In one aspect of theinvention an oligonucleotide is reversibly hybridized with apolynucleotide analyte in the presence of a 5′-nuclease under isothermalconditions. In this way the polynucleotide analyte serves as a“recognition element” to enable the 5′-nuclease to specifically cleavethe oligonucleotide to provide first and second fragments when theoligonucleotide is reversibly hybridized to the polynucleotide analyte.The first fragment comprises the 5′-end of the oligonucleotide (withreference to the intact or original oligonucleotide) and issubstantially non-hybridizable to the polynucleotide analyte and canserve as a label. The first fragment generally includes at least aportion of that part the 5′-end of the original oligonucleotide that wasnot hybridized to the polynucleotide analyte when the portion of theoligonucleotide that is hybridizable with the polynucleotide analyte isreversibly hybridized thereto. Additionally, the first fragment mayinclude nucleotides (usually, no more than 5, preferably, no more than2, more preferably, no more than 1 of such nucleotides) that are cleavedby the 5′-nuclease from the 5′-end of that portion (or sequence) of theoriginal oligonucleotide that was hybridized to the polynucleotideanalyte. Therefore, it is in the above context that the first fragmentis “substantially non-hybridizable” with the polynucleotide analyte. Thesecond fragment comprises the sequence of nucleotides at the 3′-end ofthe oligonucleotide that were reversibly hybridized to thepolynucleotide analyte minus those nucleotides cleaved by the5′-nuclease when the original oligonucleotide is reversibly hybridizedto the polynucleotide analyte. Accordingly, the second fragment is“substantially hybridizable” to the polynucleotide analyte havingresulted from that portion of the oligonucleotide that reversiblyhybridizes with the polynucleotide analyte.

As mentioned above, the 3′-end of the oligonucleotide may include one ormore nucleotides that do not hybridize with the polynucleotide analyteand may comprise a label. At least a 100-fold molar excess of the firstfragment and/or the second fragment are obtained relative to the molaramount of the polynucleotide analyte. The sequence of at least one ofthe fragments is substantially preserved during the reaction. Thepresence of the first fragment and/or the second fragment is detected,the presence thereof indicating the presence of the polynucleotideanalyte.

The 5′-nuclease is generally present in an amount sufficient to causethe cleavage of the oligonucleotide, when it is reversibly hybridized tothe polynucleotide analyte, to proceed at least half as rapidly as themaximum rate achievable with excess enzyme, preferably, at least 75% ofthe maximum rate. The concentration of the 5′-nuclease is usuallydetermined empirically. Preferably, a concentration is used that issufficient such that further increase in the concentration does notdecrease the time for the amplification by over 5-fold, preferably2-fold. The primary limiting factor generally is the cost of thereagent. In this respect, then, the polynucleotide analyte, or at leastthe target polynucleotide sequence, and the enzyme are generally presentin a catalytic amount.

The oligonucleotide that is cleaved by the enzyme is usually in largeexcess, preferably, 10⁻⁹ M to 10⁻⁵ M, and is used in an amount thatmaximizes the overall rate of its cleavage in accordance with thepresent invention wherein the rate is at least 10%, preferably, 50%,more preferably, 90%, of the maximum rate of reaction possible.Concentrations of the oligonucleotide lower than 50% may be employed tofacilitate detection′ of the fragments() produced in accordance with thepresent invention. The amount of oligonucleotide is at least as great asthe number of molecules of product desired. Usually, the concentrationof the oligonucleotide is 0.1 nanomolar to 1 millimolar, preferably, 1nanomolar to 10 micromolar. It should be noted that increasing theconcentration of the oligonucleotide causes the reaction rate toapproach a limiting value that depends on the oligonucleotide sequence,the temperature, the concentration of the target polynucleotide sequenceand the enzyme concentration. For many detection methods very highconcentrations of the oligonucleotide may make detection more difficult.

The amount of the target polynucleotide sequence that is to be copiedcan be as low as one or two molecules in a sample but generally may varyfrom about 10² to 10¹⁰, more usually from about 10³ to 10⁸ molecules ina sample preferably at least 10⁻²¹M in the sample and may be 10⁻¹⁰ to10⁻¹⁹M, more usually 10⁻¹⁴ to 10⁻¹⁹M.

In carrying out the methods in accordance with the present invention, anaqueous medium is employed. Other polar solvents may also be employed ascosolvents, usually oxygenated organic solvents of from 1-6, moreusually from 1-4, carbon atoms, including alcohols, ethers and the like.Usually these cosolvents, if used, are present in less than about 70weight percent, more usually in less than about 30 weight percent.

The pH for the medium is usually in the range of about 4.5 to 9.5, moreusually in the range of about 5.5-8.5, and preferably in the range ofabout 6-8. The pH and temperature are chosen so as to achieve thereversible hybridization or equilibrium state under which cleavage of anoligonucleotide occurs in accordance with the present invention. In someinstances, a compromise is made in the reaction parameters in order tooptimize the speed, efficiency, and specificity of these steps of thepresent method. Various buffers may be used to achieve the desired pHand maintain the pH during the determination.

Illustrative buffers include borate, phosphate, carbonate, Tris,barbital and the like. The particular buffer employed is not critical tothis invention but in individual methods one buffer may be preferredover another.

As mentioned above the reaction in accordance with the present inventionis carried out under isothermal conditions. The reaction is generallycarried out at a temperature that is near the melting temperature of theoligonucleotide:polynucleotide analyte complex. Accordingly, thetemperature employed depends on a number of factors. Usually, forcleavage of the oligonucleotide in accordance with the presentinvention, the temperature is about 35° C. to 90° C. depending on thelength and sequence of the oligonucleotide. It will usually be desiredto use relatively high temperature of 60° C. to 85° C. to provide for ahigh rate of reaction. The amount of the fragments formed depends on theincubation time and temperature. In general, a moderate temperature isnormally employed for carrying out the methods. The exact temperatureutilized also varies depending on the salt concentration, pH, solventsused, and the length of and composition of the target polynucleotidesequence as well as the oligonucleotide as mentioned above.

One embodiment of the invention is depicted in FIG. 1. OligonucleotideOL is combined with polynucleotide analyte PA having targetpolynucleotide sequence TPS and with a 5′-nuclease, which can be, forexample, a Taq polymerase. In this embodiment OL is labeled (*) withinwhat is designated the first fragment, produced upon cleavage of theoligonucleotide in accordance with the present invention. OL in thisembodiment usually is at least 10 nucleotides in length, preferably,about 10 to 50 nucleotides in length, more preferably, 15 to 30 or morenucleotides in length. In general, the length of OL should be sufficientso that a portion hybridizes with TPS, the length of such portionapproximating the length of TPS. In this embodiment the length of OL ischosen so that the cleavage of no more than 5, preferably, no more than1 to 3, more preferably, 1 to 2 nucleotides, therefrom results in twofragments. The first fragment, designated LN, is no more than 5nucleotides in length, preferably, 1 to 3 nucleotides in length, morepreferably, 1 to 2 nucleotides in length and the second fragment,designated DOL, is no more than 5, preferably, no more than 1 to 3, morepreferably, no more than 1 to 2, nucleotides shorter than the length ofOL.

As shown in FIG. 1, OL hybridizes with TPS to give duplex I. Thehybridization is carried out under isothermal conditions so that OL isreversibly hybridized with TPS. OL in duplex I is cleaved to give DOLand LN, wherein LN includes a labeled nucleotide (*). In the embodimentdepicted in FIG. 1, DOL is the complement of TPS except for thenucleotides missing at the 5′-end. Since during the course of theisothermal reaction the 5′-end of PA may be cleaved at or near the5′-end of TPS, DOL may also have 0 to 5 nucleotides at its 3′-end thatoverhang and cannot hybridize with the residual portion of TPS. Theisothermal conditions are chosen such that equilibrium exists betweenduplex I and its single stranded components, namely, PA and OL. Uponcleavage of OL within duplex I, an equilibrium is also establishedbetween duplex I and its single stranded components, PA and DOL. SinceOL is normally present in large excess relative to the amount of DOLformed in the reaction, there are usually many more duplexes containingOL than DOL. The reaction described above for duplex I continuouslyproduces additional molecules of DOL.

The reaction is allowed to continue until a sufficient number ofmolecules of DOL and LN are formed to permit detection of the labeled LN(LN*) and, thus, the polynucleotide analyte. In this way theenzyme-catalyzed cleavage of nucleotides from the 5′-end of OL ismodulated by and, therefore, related to the presence of thepolynucleotide analyte. Depending on the amount of PA present, asufficient number of molecules for detection can be obtained where thetime of reaction is from about 1 minute to 24 hours. Preferably, thereaction can be carried out in less than 5 hours. As a matter ofconvenience it is usually desirable to minimize the time period as longas the requisite of number of molecules of detectable fragment isachieved. In general, the time period for a given degree of cleavage canbe minimized by optimizing the temperature of the reaction and usingconcentrations of the 5′-nuclease and the oligonucleotide that providereaction rates near the maximum achievable with excess of thesereagents. Detection of the polynucleotide analyte is accomplishedindirectly by detecting the label in fragment LN*. Alternatively, DOLmay be detected, for example, by using the label as a means ofseparating LN* and OL from the reaction mixture and then detecting theresidual DOL.

Detection of the labeled fragment is facilitated in a number of ways.For example, a specific pair member such as biotin or a directlydetectable label such a fluorescein can be used. The low molecularweight LN* can be separated by electrophoresis, gel exclusionchromatography, thin layer chromatography ultrafiltration and the likeand detected by any convenient means such as a competitive binding assayor direct detection of the label. Alternatively, the oligonucleotide canbe labeled within the second (DOL) fragment with a specific bindingmember such as a ligand, a small organic molecule, a polynucleotidesequence or a protein, or with a directly detectable label such as adirectly detectable small organic molecules, e.g., fluorescein, asensitizer, a coenzyme and the like. Detection will then depend ondifferentiating the oligonucleotide with labels on both ends from singlylabeled fragments where one labeled end has been cleaved. In this caseit is desirable to label one end of OL with a specific binding memberthat facilitates removal of OL and the fragment retaining the label byusing a complementary sbp member bound to a support. The residuallabeled fragments bearing the other label are then detected by using amethod appropriate for detecting that label.

One method for detecting nucleic acids is to employ nucleic acid probes.Other assay formats and detection formats are disclosed in U.S. patentapplication Ser. Nos. 07/229,282 and 07/399,795 filed Jan. 19, 1989, andAug. 29, 1989, respectively, U.S. patent application Ser. No. 07/555,323filed Jul. 19, f990, U.S. patent application Ser. No. 07/555,968 andU.S. patent application Ser. No. 07/776,538 filed Oct. 11, 1991, whichhave been incorporated herein by reference. Examples of particularlabels or reporter molecules and their detection can be found in U.S.patent application Ser. No. 07/555,323 filed Jul. 19, 1990, the relevantdisclosure of which is incorporated herein by reference.

Detection of the signal will depend upon the nature of the signalproducing system utilized. If the label or reporter group is an enzyme,additional members of the signal producing system include enzymesubstrates and so forth. The product of the enzyme reaction ispreferably a luminescent product, or a fluorescent or non-fluorescentdye, any of which can be detected spectrophotometrically, or a productthat can be detected by other spectrometric or electrometric means. Ifthe label is a fluorescent molecule, the medium can be irradiated andthe fluorescence determined. Where the label is a radioactive group, themedium can be counted to determine the radioactive count.

Another embodiment of the present invention is depicted in FIG. 2.Oligonucleotide OL′ has a first portion or sequence SOL1 that is nothybridized to TPS′ and a second portion or sequence SOL2 that ishybridized to TPS′. OL′ is combined with polynucleotide analyte PA′having target polynucleotide sequence TPS′ and with a 5′-endonuclease(5′-endo), which can be, for example, Taq DNA polymerase and the like.OL′ and 5′-endo are generally present in concentrations as describedabove. In the embodiment of FIG. 2, OL′ is labeled (*) within thesequence SOL1 wherein SOL1 may intrinsically comprise the label or maybe extrinsically labeled with a specific binding member or directlydetectable labeled. The length of SOL2 is as described in the embodimentof FIG. 1. In general, the length of SOL2 should be sufficient tohybridize with TPS′, usually approximating the length of TPS′. SOL1 maybe any length as long as it does not substantially interfere with thecleavage of OL′ and will preferably be relatively short to avoid suchinterference. Usually, SOL1 is about 1 to 100 nucleotides in length,preferably, 8 to 20 nucleotides in length.

In this embodiment the cleavage of SOL1 from SOL2 results in twofragments. Cleavage in SOL2 occurs within 5 nucleotides of the bondjoining SOL1 and SOL2 in OL′. The exact location of cleavage is notcritical so long as the enzyme cleaves OL′ only when it is bound toTPS′. The two fragments are designated LNSOL1 and DSOL2. LNSOL1 iscomprised of the 5′-end of OL′ and DSOL2 is comprised of the 3′-end ofOL′. The sequence of at least one of LNSOL1 and DSOL2 remainssubstantially intact during the cleavage reaction. As shown in FIG. 2,SOL2 of OL′ hybridizes with TPS′ to give duplex I′. The hybridization iscarried out under isothermal conditions so that OL′ is reversiblyhybridized with TPS′. OL′ in duplex I′ is cleaved to give DSOL2 andLNSOL1, the latter of which comprises a label. In the embodimentdepicted in FIG. 2, DSOL2 is the complement of TPS′ except for anynucleotides missing at the 5′-end thereof as a result of the cleavage ofthe cleavage reaction and any nucleotides appended to the 3′-end of OL′(not shown in FIG. 2) that do not hybridize with TPS′.

The isothermal conditions are chosen such that equilibrium existsbetween duplex I′ and its single stranded components, i.e., PA′ and OL′.Upon cleavage of OL′ within duplex I′ and equilibrium is alsoestablished between duplex I′ and its single stranded components, PA′and DSOL2. Since OL′ is normally present in large excess relative to theamount of DSOL2 formed in the reaction, there are usually many moreduplexes containing OL′ than DSOL2. The reaction described above forduplex I′ continuously produces molecules of DSOL2 and LNSOL1. Thereaction is allowed to continue until a sufficient number of moleculesof DSOL2 and LNSOL1 are formed to permit detection of one or both ofthese fragments. In this way the enzyme-catalyzed cleavage of LNSOL1from the 5′-end of the portion of OL′ hybridized to PA′ is modulated by,and therefore related to, the presence of the polynucleotide analyte.The reaction parameters and the detection of DSOL2 and/or LNSOL1 aregenerally as described above for the embodiment of FIG. 1.

Various ways of controlling the cleavage of the oligonucleotide can beemployed. For example, the point of cleavage can be controlled byintroducing a small organic group, such as biotin, into the nucleotideat the 5′-terminus of OL′ or the nucleotide in SOL2 that is at thejunction of SOL2 and SOL1.

An embodiment using a second oligonucleotide is depicted in FIG. 3. Thesecond oligonucleotide (OL2) hybridizes-to a site TPS2 on PA″ that lies3′ of the site of hybridization (TPS1) of the sequence SOL2″ of thefirst oligonucleotide, namely, OL″. In the embodiment shown OL2 fullyhybridizes with TPS2. This is by way of example and not limitation. Thesecond oligonucleotide can include nucleotides at its 5′end that are nothybridizable with the target polynucleotide sequence, but its 3′-end ispreferably hybridizable. Preferably, OL2 binds to a site (TPS2) that iscontiguous with the site to which SOL2″ hybridizes (TPS1). However, itis within the purview of the present invention that the secondoligonucleotide hybridize with PA″ within 1 to 5 nucleotides,preferably, 1 nucleotide, of the site to which SOL2″ hybridizes. Thesecond oligonucleotide, OL2, is usually at least as long as, andpreferably longer than, SOL2″, preferably, at least 2 nucleotides longerthan SOL2″. In general, the second oligonucleotide is about 20-100nucleotides in length, preferably, 30-80 nucleotides in length dependingon the length of SOL2″. Normally, the second oligonucleotide is chosensuch that it dissociates from duplex I″ at a higher temperature thanthat at which OL″ dissociates, usually at least 3° C., preferably, atleast 5° C. or more higher.

The presence of OL2 in duplex I″ can effect the site of cleavage of OL″.In particular, when OL2 binds to PA″ that is not contiguous with theSOL2″ site of hybridization, the cleavage site may be shifted one ormore nucleotides.

The concentration of the second oligonucleotide employed in thisembodiment is usually at least 1 picomolar, but is preferably above 0.1nanomolar to facilitate rapid binding to PA″, more preferably, at least1 nanomolar to 1 micromolar. In accordance with the embodiment of FIG.3, OL″ in duplex I″ is cleaved by 5′-endo to give DSOL2″ and LNSOL1″.The reaction is permitted to continue until the desired number ofmolecules of labeled fragment are formed. The reaction parameters anddetection of DSOL2″ and/or LNSOL1″ are similar to those described abovefor the embodiment of FIG. 1.

In general and specifically in any of the embodiments of FIGS. 1 to 3above, the 3′-end of the first oligonucleotide, for example, OL, OL′ andOL″, may have one or more nucleotides that do not hybridize with thetarget polynucleotide sequence and can serve as a label but need not doso.

It is also within the purview of the present invention to employ asingle nucleoside triphosphate in any of the above embodiments,depending on the particular 5′-endonuclease chosen for the abovecleavage. The decision to use a nucleoside triphosphate and the choiceof the nucleoside triphosphate are made empirically based on its abilityto accelerate the reaction in accordance with the present invention. Thenucleoside triphosphate is preferably one that cannot be incorporatedinto the first oligonucleotide as a consequence of the binding of theoligonucleotide to the target polynucleotide sequence. In thisparticular embodiment the added nucleoside triphosphate is present in aconcentration of 1 micromolar to 10 millimolar, preferably, 10micromolar to 1 millimolar, more preferably, 100 micromolar to 1millimolar. It is also within the purview of the present invention toutilize the added nucleoside triphosphate to chain extend the3′-terminus of the second oligonucleotide to render it contiguous withthe site on the target polynucleotide sequence at which the firstoligonucleotide hybridizes. In this approach the second oligonucleotideserves as a polynucleotide primer for chain extension. In addition, thenucleoside triphosphate is appropriately selected to accomplish suchchain extension and the 5′-nuclease is selected to also havetemplate-dependent nucleotide polymerase activity. In any event such anapproach is primarily applicable to the situation where the site ofbinding of this second oligonucleotide, TPS2, is separated from the siteof binding of the first oligonucleotide, TPS1, by a sequence of one ormore identical bases that are complementary to the added nucleotidetriphosphate.

In the embodiment of FIG. 3 the mixture containing PA″, OL″, the secondoligonucleotide OL2 and the nucleoside triphosphate is incubated at anappropriate isothermal temperature at which OL″ and PA″ are inequilibrium with duplex I″ wherein most of the molecules of PA″ andduplex I″ are hybridized to OL2. During the time when a molecule of OL″is bound to PA″, the 5′-endo causes the cleavage by hydrolysis of OL″ inaccordance with the present invention. When the remaining portion ofcleaved oligonucleotide (DSOL2″) dissociates from PA″, an intactmolecule of OL″ becomes hybridized, whereupon the process is repeated.

In one experiment in accordance with the above embodiment, incubationfor 3 hours at 72° C. resulted in the production of over 10¹² moleculesof DSOL2″ and LNSOL1″, which was over 10⁴ increase over the number ofmolecules of PA″ that was present initially in the reaction mixture. OL″was labeled with a ³²P-phosphate at the 5′-terminus. The cleaved productLNSOL1″ was detected by applying the mixture to an electrophoresis geland detecting a band that migrated more rapidly than the band associatedwith OL″. The appearance of this band was shown to be associated withthe presence and amount of PA″ where a minimum of 10⁸ molecules of PA″was detected.

Alternative approaches for detection of LNSOL1″ and/or DSOL2″ may alsobe employed in the above embodiment. For example, in one approach biotinis attached to any part of SOL2″ that is cleaved from OL″ by the5′-endonuclease. The fragment DSOL2″ and OL″ containing the biotin areseparated from LNSOL1″, for example, by precipitation with streptavidinand filtration. The unprecipitated labeled fragment LNSOL1″ is thendetected by any standard binding assay, either without separation(homogeneous) or with separation (heterogeneous) of any of the assaycomponents or products.

Homogeneous immunoassays are exemplified by enzyme multipliedimmunoassay techniques (“EMIT”) disclosed in Rubenstein, et al., U.S.Pat. No. 3,817,837, column 3, line 6 to column 6, line 64;immunofluorescence methods such as those disclosed in Ullman, et al.,U.S. Pat. No. 3,996,345, column 17, line 59 to column 23, line 25;enzyme channeling techniques such as those disclosed in Maggio, et al.,U.S. Pat. No. 4,233,402, column 6, line 25 to column 9, line 63; andother enzyme immunoassays such as the enzyme linked immunosorbant assay(“ELISA”) are discussed in Maggio, E. T. supra. Exemplary ofheterogeneous assays are the radioimmunoassay, disclosed in Yalow, etal., J. Clin. Invest. 39:1157 (1960). The above disclosures are allincorporated herein by reference. For a more detailed discussion of theabove immunoassay techniques, see “Enzyme-Immunoassay,” by Edward T.Maggio, CRC Press, Inc., Boca Raton, Fla., 1980. See also, for example,U.S. Pat. Nos. 3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987;3,867,517; 3,901,654; 3,935,074; 3,984,533; 3,996,345; and 4,098,876,which listing is not intended to be exhaustive.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive assay formats are disclosed in Davalian, et al., U.S.Pat. No. 5,089,390, column 14, line-25 to column 15, line 9,incorporated herein by reference. A typical non-competitive assay is asandwich assay disclosed in David, et al., U.S. Pat. No. 4,486,530,column 8, line 6 to column 15, line 63, incorporated herein byreference.

Another binding assay approach involves the luminescent immunoassaydescribed in U.S. Ser. No. 07/704,569, filed May 22, 1991 entitled“Assay Method Utilizing Induced Luminescence”, which disclosure isincorporated herein by reference.

As a matter of convenience, predetermined amounts of reagents employedin the present invention can be provided in a kit in packagedcombination. A kit can comprise in packaged combination (a) a firstoligonucleotide having the characteristic that, when reversiblyhybridized to a portion of a polynucleotide to be detected, it isdegraded under isothermal conditions by a 5′-nuclease to provide (i) afirst fragment that is substantially non-hybridizable to thepolynucleotide and (ii) a second fragment that is 3′ of the firstfragment and is substantially hybridizable to the polynucleotide, (b) asecond oligonucleotide having the characteristic of at least a portionthereof hybridizing to a site on the polynucleotide that is 3′ of thesite at which the first oligonucleotide hybridizes wherein thepolynucleotide is substantially fully hybridized to such portion of thesecond oligonucleotide under isothermal conditions, and (c) the above5′-nuclease. The kit can further comprise a single nucleosidetriphosphate.

The above kits can further include members of a signal producing systemand also various buffered media, some of which may contain one or moreof the above reagents. The above kits can also include a writtendescription of one or more of the methods in accordance with the presentinvention for detecting a polynucleotide analyte.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents which substantiallyoptimize the reactions that need to occur during the present method andto further substantially optimize the sensitivity of any assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay inaccordance with the present invention. Each reagent can be packaged inseparate containers or some reagents can be combined in one containerwhere cross-reactivity and shelf life permit.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples. Temperatures are in degrees centigrade (° C.) and parts andpercentages are by weight, unless otherwise indicated.

Example 1

A single stranded target DNA (2×10⁸ molecules) (M13mp19 from Gibco, BRL,Bethesda, Md.) (the “target DNA”) was combined with a 5′³²P-labeledoligonucleotide probe, Probe 1, (10 uM) (5′CGT-GGG-AAC-AAA-CGG-CGG-AT3′(SEQ ID NO:1) synthesized on a Pharmacia Gene Assembler (PharmaciaBiotech, Piscataway, N.J.), an unlabeled oligonucleotide, Probe 2, (1uM) (5′TTC-ATC-AAC-ATT-AAA-TGT-GAG-CGA-GTA-ACA-ACC-CGT-CGG-ATT-CTC3′(SEQ ID NO:2) synthesized on a Pharmacia Gene Assembler (PharmaciaBiotech), and 7.5 units of AmpliTaq DNA polymerase (from Perkin-ElmerCorporation, Norwalk, N.J.) in 50 uL of buffer (10 mM Tris-HCl, pH 8.5,50 mM KCl, 7.5 mM MgCl₂, 100 uM DATP). Probe 1 was a 20-baseoligonucleotide that was fully complementary to the target DNA and had alabel on the 5′-nucleotide. Probe 2, the unlabeled probe, was designedto anneal to the target DNA 3′ to, and contiguous with, the site atwhich the labeled probe annealed to the target DNA. The dATP was shownto enhance the rate of cleavage by the polymerase. However, good resultswere obtained in the absence of dATP.

The reaction mixture was incubated at 72° C. and accumulation ofproduct, a mononucleotide, namely, 5′³²P-C-OH, was determined byvisualization using autoradiography following polyacrylamide gelelectrophoresis. The fold of amplification was determined by liquidscintillation spectrometry of excised reaction products. A 10⁵ foldamplification was observed.

The above reaction protocol was repeated using, in place of Probe 1, alabeled probe, Probe 3, (5′TCG-TGG-GAA-CAA-ACG-GCG-GAT3′ (SEQ ID NO:3)prepared using a Pharmacia Gene Assembler) that had 21 nucleotides withone base at the 5′-end that was not complementary, and did not hybridizewith, the target DNA. The product of this reaction was a dinucleotide,namely, 5′³²P-TC-OH (SEQ ID NO:4), that represented a 10⁵-foldamplification.

The above reaction protocol was repeated with different temperatures anddifferent concentrations of reagents. All of the reactions, includingthose mentioned above, were carried out for a period of 3 hours. Thefollowing table summarizes the reagents and reaction parameters and theresults obtained during the optimization procedure. Probe Target TaqTemp° Fold Probe (μM) number (units) C. Conditions amplification 1 1 10¹⁰ 2.5 72 buffer as 8.8 × 10² described; 1.5 mM MgCl₂ 1 10⁹ | | | 1.8× 10³ 1 10⁸ ↓ | | N.D.* 1 10⁹ 7.5 | ↓ 2.0 × 10³ 1 10⁹ | | add dATP 1.4 ×10³ (100 μM) 1 10⁸ | | | 1.0 × 10⁴ 10 10⁹ | | | 1.4 × 10⁴ 10 10⁸ | | ↓3.6 × 10⁴ 1 10⁹ | | increase MgCl² 9.7 × 10³ (7.5 mM) 1 10⁸ | | | 1.2 ×10⁴ 1 10⁹ | | | 9.3 × 10³ 1 10⁸ | | | 2.8 × 10⁴ 1 10⁷ | ↓ | N.D.* 10 10⁹| 74 | 3.7 × 10⁴ 10 10⁸ | | | 1.1 × 10⁵ 10 10⁷ | ↓ | N.D.* 3 1 10⁹ | 72| 9.9 × 10³ 1 10⁸ | | | 2.6 × 10⁴ 1 10⁷ | ↓ | N.D.* 10 10⁹ | 74 | 4.6 ×10⁴ 10 10⁸ | | | 1.0 × 10⁵ 10 10⁷ ↓ ↓ ↓ N.D.**N.D. = not detected

Example 2

The reaction protocol described in Example 1 was repeated using thefollowing probes in place of Probe 1 or Probe 3:

-   -   Probe 4: 5′TTA-TTT-CGT-GGG-AAC-AAA-CGG-CGG-AT3′ (SEQ ID NO:5)        (from Oligos Etc., Inc., Wilsonville, Oreg.). Probe 4 had 26        nucleotides with six nucleotides at its 5′ -end that were not        complementary, nor hybridizable with, the target DNA. Probe 4        was present in a concentration of 1 micromolar. The product of        this reaction was an intact seven nucleotide fragment, namely,        5′³²P-TTATTTC-OH (SEQ ID NO:6), that represented a 1.5×10⁴-fold        amplification.    -   Probe 5: 5′GAT-TAG-GAT-TAG-GAT-TAG-TCG-TGG-GAA-CAA-ACG-GCG-GAT3′        (SEQ ID NO:7) was prepared using a Pharmacia Gene assembler and        had 39 nucleotides with 19 nucleotides at its 5′-end that were        not complementary and did not hybridize with the target DNA. The        product of this reaction was an intact 20 nucleotide fragment,        namely, 5′³²P-GAT-TAG-GAT-TAG-GAT-TAG-TC-OH (SEQ ID NO:8), that        represented a 1.5×10⁴-fold amplification.

In repeating the above reactions in the absence of Probe 2, product wasobserved but the intensity of the spot on the polyacrylamide gel wassignificantly less than in the presence of Probe 2. Similar results werealso observed where a 1 nucleotide space existed between the 3′-end ofProbe 2 and the second probe when both probes were hybridized to thetarget DNA.

Example 3

The reaction protocol described in Example 1 was repeated using 2×10⁹target molecules and Probe 5 (see Example 2) at a concentration of 1micromolar in place of Probe 1. The reactions were conducted for threehours at a temperature of 72° C. using one of six different DNApolymerases, namely, AmpliTaq DNA polymerase, Replitherm(™) DNApolymerase (Epicentre), Tfl (™) DNA polymerase (Epicentre), Ultra Therm(™) DNA polymerase (Bio/Can Scientific), Thermalase Tbr(™) DNApolymerase (Amresco) and Panozyme(™) DNA polymerase. The product of thereaction was a 20-nucleotide fragment (see Example 2) The following is asummary of the results obtained. Enzyme Fragment (picomoles) AmpliTaq 32Replitherm 18 Tfl 5 Ultra Therm 27 Tbr 16 Panozyme 25

The above experiments demonstrate that detectable cleavage products weregenerated in a target-specific manner at a single temperature usingenzymes having 5′-nuclease activity and a labeled oligonucleotide. Theaccumulation of product was enhanced by the presence of a secondoligonucleotide that was longer than the first labeled oligonucleotideand that was annealed to the target polynucleotide sequence 3′ of thesite of hybridization of the first labeled oligonucleotide. Thereactions were carried out at temperatures very close to the meltingtemperature (Tm) of the labeled oligonucleotide with the targetpolynucleotide sequence.

The above discussion includes certain theories as to mechanisms involvedin the present invention. These theories should not be construed tolimit the present invention in any way, since it has been demonstratedthat the present invention achieves the results described.

The above description and examples fully disclose the inventionincluding preferred embodiments thereof. Modifications of the methodsdescribed that are obvious to those of ordinary skill in the art such asmolecular biology and related sciences are intended to be within thescope of the following claims.

1-34. (canceled)
 35. A kit for detection of a polynucleotide comprisingin packaged combination: (a) a first oligonucleotide having thecharacteristic that, when reversibly hybridized under isothermalconditions to at least a portion of the polynucleotide, it is degradedby a 5′-nuclease to provide (i) a first fragment that is substantiallynon-hybridizable to the polynucleotide and (ii) a second fragment thatis 3′ of the first fragment in the first oligonucleotide and issubstantially hybridizable to the polynucleotide, wherein the isothermalconditions are at or near the melting temperature of a duplex comprisingthe second fragment hybridized to the polynucleotide, and (b) a secondoligonucleotide having the characteristic of hybridizing to a site onthe polynucleotide that is separated by no more than five nucleotidesfrom the 3′-end of the site at which the second fragment of the firstoligonucleotide hybridizes, wherein the polynucleotide is substantiallyfully hybridized to the second oligonucleotide at said meltingtemperature.
 36. The kit of claim 35 wherein the second oligonucleotidehybridizes to a site on the polynucleotide that is separated by no morethan one nucleotide from the 3′-end of the site at which the secondfragment of the first oligonucleotide hybridizes.
 37. The kit of claim35, wherein the second oligonucleotide hybridizes to a site on thepolynucleotide that is contiguous with the 3′-end of the site at whichthe second fragment of the first oligonucleotide hybridizes.
 38. The kitof claim 35, wherein the second oligonucleotide dissociates from theduplex at a temperature at least 3° C. higher than the temperature ofsaid melting point.
 39. The kit of claim 35, wherein the secondoligonucleotide dissociates from the duplex at a temperature at least 5°C. higher than the temperature of said melting point.
 40. The kit ofclaim 35, wherein the second oligonucleotide is at least as long as thesecond fragment of the first oligonucleotide.
 41. The kit of claim 35,wherein the second oligonucleotide is at least 2 nucleotides longer thanthe second fragment of the first oligonucleotide.
 42. The kit of claim35, further comprising a label.
 43. The kit claim 35, wherein the firstfragment of the first oligonucleotide comprises a label.
 44. The kit ofclaim 35, further comprising a buffering media.
 45. The kit of claim 35,further comprising a 5′-nuclease.
 46. The kit of claim 45, wherein the5′-nuclease comprises a 5′-endonuclease.
 47. The kit of claim 45,wherein the 5′-nuclease comprises a Taq polymerase.